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If your question is not answered here, please email us at:  ${email.software} # 10x GenomicsChromium Genome & Exome # Reference Support Long Ranger algorithms are tuned and optimized for human haplotype phasing and structural variant calling, and 10x Genomics provides pre-built hg19 and b37-style reference packages for use with the pipeline. The pre-built references have the following characteristics: • Human GRCh37 build • Two variants: • hg19/UCSC-style chromosome naming convention ("chr1") • b37/1000 Genomes-style chromosome naming convention ("1") Use of the pre-built references is strongly recommended unless you have specific requirements that match one of the compatible use cases below: ## Compatible Use Cases Long Ranger supports the use of customer-generated references for the following scenarios: • Small numbers of additional contigs (e.g. decoy and viral sequences) ## Not Compatible The following scenarios are not currently supported by Long Ranger: • Incomplete human assemblies with large numbers of contigs (> 250) • Non-human genomes ## Making a Reference Package There are 3 steps to construct a Long Ranger-compatible reference. You must have version 1.1.1 or later of both Long Ranger and Loupe. ### 1. Index your FASTA To create a reference, run the longranger mkref command on your FASTA file. The contigs in your FASTA must meet the compatibility requirements above. $ longranger mkref hsapiens-asm19.fasta
... indexing may take over an hour ...
$ls refdata-hsapiens-asm19 fasta/ genes/ genome regions/ snps/  This utility copies your FASTA, indexes it in several formats, and outputs a folder named refdata-<fasta_name>. ### 2. SV Calling Blacklist At this point, the reference folder created by longranger mkref is usable by Long Ranger, but it is strongly recommended that you also include a region blacklist for structural variant calling. The blacklist is used by the SV algorithm to reduce false positives due to gaps in the reference, known or putative assembly issues such as unplaced contigs, and highly polymorphic regions. For hg19 references, we provide pre-built blacklist files that you can simply copy into your reference. Follow the instructions below, depending on the naming convention of your reference: hg19 Convention ("chr1") $ cd refdata-hsapiens-asm19
$cd regions$ wget https://cf.10xgenomics.com/supp/genome/hg19/sv_blacklist.bed
$wget https://cf.10xgenomics.com/supp/genome/hg19/segdups.bedpe  b37 Convention ("1") $ cd refdata-hsapiens-asm19
$cd regions$ wget https://cf.10xgenomics.com/supp/genome/b37/sv_blacklist.bed
$wget https://cf.10xgenomics.com/supp/genome/b37/segdups.bedpe  For all other references, follow these instructions to create custom blacklist files. You may also limit variant calling to a subset of contigs in the reference by including a primary_contigs.txt file in the fasta directory. This is a contig whitelist, as opposed to a region blacklist. You can see an example in the fasta folder of the refdata-hg19-2.0.0 reference. It may be useful to exclude some contigs from the whitelist, such as decoys or alts. ### 3. Genes/Exons File for Loupe To enable the display of the genes and exons tracks in the Loupe genome browser, download our gene annotations file into your reference. The annotation source can be found at ENSEMBL. This file will work regardless of the naming convention of your reference. $ cd refdata-hsapiens-asm19
$cd genes$ wget https://cf.10xgenomics.com/supp/genome/gene_annotations.gtf.gz


This step is optional, but if you omit this file, you will not be able to search by gene name in Loupe, or see the genes and exons tracks in the Loupe Haplotype view. Loupe will accept any GTF subject to the following requirements:

• Loupe only considers records with a "feature" of "gene" or "exon"; other feature types will be ignored.
• Loupe requires all records to have a "gene_id" and "gene_name" tag.
• Exons must have the same gene_id tag as their respective gene.
• The file must be compressed in gzip format.

### 4. Confirm Contents

If you have followed the steps above correctly, your reference folder should now contain the following files: